A diverse proteome is present and enzymatically active in metabolite extracts

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

Rachel (Rae) J. House, Molly T. Soper-Hopper, Michael P. Vincent, Abigail E. Ellis, Colt D. Capan, Zachary B. Madaj, Emily Wolfrum, Christine N. Isaguirre, Carlos D. Castello, Amy B. Johnson, Martha L. Escobar Galvis, Kelsey S. Williams, Hyoungjoo Lee, Ryan D. Sheldon